Browsing by Author "Rangan, Rajiv"
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Item ADAM19 and ADAMTS4 expression in Human Optic Nerve Head Astrocytes(2024-03-21) Easo, Tony; Rangan, Rajiv; Tovar-Vidales, TaraPurpose: Glaucoma is characterized by the degeneration and death of retinal ganglion cells and their axons causing irreversible blindness. Various risk factors contribute to glaucoma onset, including intraocular pressure (IOP), age, and family history. In glaucoma, the primary site of damage is the optic nerve head (ONH). ONH astrocytes, a major cell type in the LC, are believed to play a significant role in the pathological remodeling of extracellular matrix (ECM) during glaucoma. Glaucoma patients have increased levels of transforming growth factor beta 2 (TGFβ2) in their aqueous humor, trabecular meshwork and ONH. TGFβ2 is a profibrotic cytokine known to induce the synthesis and deposition of ECM. Previously, we performed RNA sequencing of ONH astrocytes in which disintegrin and metalloproteinases (ADAMs) and ADAM with thrombospondin motifs (ADAMTS) were significantly dysregulated with TGFβ2 treatment compared to controls. ADAMs and ADAMTS4 influence cell phenotype by affecting cell adhesion, migration, proteolysis, and signaling pathways. The purpose of the present study was to determine a) if ONH astrocytes express ADAM19 and ADAMTS4 in ONH astrocytes and b) determine if TGFβ2 regulates ADAM19 and ADAMTS4 expression in ONH astrocytes. Methods: Primary human ONH astrocyte cell strains (n=3) were treated with TGFβ2 (5ng/ml) or with a vehicle control for 48 hours. The effects of TGFβ2 on ADAM19 and ADAMTS4 were determined by qPCR and western blots using primary human ONH astrocyte cell cultures. Results: ADAM19 and ADAMTS4 were expressed in ONH astrocytes. Treatment with TGFβ2 significantly increased mRNA levels of ADAM19 and ADAMTS4. However, western blot analysis showed no significant change in ADAM19 protein expression compared to control, while ADAMTS4 was increased with TGFβ2 compared to control. Conclusions: TGFβ2 modulated the expression of ADAM19 and ADAMTS4 in ONH astrocytes. ADAM19 and ADAMTS4 may contribute to the pathogenic remodeling of the ONH in glaucoma. While further studies are needed, this research aims to shed light on the intricate mechanisms underlying glaucoma pathogenesis.Item Increased Fibronectin Serotonylation in Stretched Optic Nerve Head Astrocytes(2023) Rangan, Rajiv; Clark, Abbot; Tovar-Vidales, TaraPurpose: Elevated intraocular pressure contributes to glaucomatous optic nerve degeneration by inducing biomechanical stress at the optic nerve head (ONH), especially in the lamina cribrosa (LC). ONH astrocytes (ONHA) in the LC respond to biomechanical signals through extracellular matrix (ECM) remodeling activities, promoting tissue fibrosis and damage to retinal ganglion cell axons. The enzyme transglutaminase 2 (TG2) plays a role in ECM remodeling, in part, due to its ability to post-translationally modify and cross-link ECM proteins. A unique post-translational modification mediated by TG2 is "serotonylation” - the transamidation of the monoamine serotonin (5-hydroxytryptamine, 5HT) to glutamine residues on proteins. It is speculated that serotonylation contributes to fibrotic tissue remodeling, but this process has not been studied in ocular tissues or in primary glial cells. In this study, we examined changes in the serotonylation of fibronectin (FN; a major ECM glycoprotein) by ONHA after exposure to cyclic stretch. Methods: Primary human ONHA strains (n=3) were exposed to 0-12% cyclic stretch for 24h using a FlexCell FX-6000 system. Cell lysates and conditioned medium samples were collected from stretched and control cells. Serotonylation was assessed by probing for serotonin in samples of FN immunoprecipitated out of conditioned media. Protein levels for potential extra- and intra-cellular mediators of serotonylation were examined using western blotting of concentrated conditioned medium samples and cell lysates, respectively. Results: Serotonylated fibronectin was detected in ONHA. Exposure to stretch increased the amount of fibronectin that was serotonylated by 2.49-fold (p=0.0080). After stretching, extracellular FN levels were not changed. Extracellular TG2 levels were increased by 3.76-fold (p=0.0004). In cell lysates, post-stretch levels of both FN and TG2 were decreased by 5.56-fold (p=0.0181) and 2.51-fold (p=0.0441), respectively. Additionally, serotonin 2A and 2C (5HT2A, 5HT2C) receptor levels were unchanged, and serotonin transporter (SERT) levels decreased by 2.94-fold (p=0.0297). Conclusions: Increased FN serotonylation by TG2 is observed in ONHA after exposure to 24h of 0-12% cyclic stretch. Serotonylation may promote increased FN-crosslinking and fibrotic ECM remodeling, an important feature of glaucomatous pathology. The secreted TG2 – which was elevated in response to stretch – is likely to be the primary mediator of this increased serotonylation. The observed decrease in SERT may lead to increased extracellular 5HT levels, which increases the substrate availability for TG2-mediated serotonylation. Though unchanged, activity at the 5HT2A/C G-coupled protein receptors could increase the availability of intracellular calcium required for TG2 activity. Future experiments will be focused on furthering our understanding of how these proteins may interact to promote serotoynlation.Item Mirna Expression in Glaucomatous and TGFbeta2 Treated Lamina Cribrosa Cells(MDPI, 2021-06-08) Lopez, Navita N.; Rangan, Rajiv; Clark, Abbot F.; Tovar-Vidales, TaraGlaucoma is a group of optic neuropathies that leads to irreversible vision loss. The optic nerve head (ONH) is the site of initial optic nerve damage in glaucoma. ONH-derived lamina cribrosa (LC) cells synthesize extracellular matrix (ECM) proteins; however, these cells are adversely affected in glaucoma and cause detrimental changes to the ONH. LC cells respond to mechanical strain by increasing the profibrotic cytokine transforming growth factor-beta 2 (TGFbeta2) and ECM proteins. Moreover, microRNAs (miRNAs or miR) regulate ECM gene expression in different fibrotic diseases, including glaucoma. A delicate homeostatic balance between profibrotic and anti-fibrotic miRNAs may contribute to the remodeling of ONH. This study aimed to determine whether modulation of miRNAs alters the expression of ECM in human LC cells. Primary human normal and glaucoma LC cells were grown to confluency and treated with or without TGFbeta2 for 24 h. Differences in expression of miRNAs were analyzed using miRNA qPCR arrays. miRNA PCR arrays showed that the miR-29 family was significantly decreased in glaucomatous LC cell strains compared to age-matched controls. TGFbeta2 treatment downregulated the expression of multiple miRNAs, including miR-29c-3p, compared to controls in LC cells. LC cells transfected with miR-29c-3p mimics or inhibitors modulated collagen expression.Item miRNA Profiling of Human Optic Nerve Head Astrocytes Exposed to Cyclic Stretch(2021) Rangan, Rajiv; Tovar-Vidales, TaraPurpose: Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, a leading cause of irreversible blindness involving the progressive loss of retinal ganglion cells (RGCs) and their axons. Elevated IOP induces biomechanical aberrations within ocular tissues – including the transmission of biomechanical stretch through the lamina cribrosa (LC) region of the optic nerve head (ONH), the site where RGC axon damage first occurs. LC cells and ONH astrocytes (ONHA), the primary cells of the LC, respond to stretch in a manner that promotes pathological extracellular matrix (ECM) remodeling and mechanical damage of RGCs within the ONH. A complex set of molecular mechanisms regulate ECM remodeling. Part of this regulation may involve microRNAs (miRNAs), small RNA molecules that can indirectly inhibit gene expression by binding messenger RNA. miRNA dysregulation may contribute to ECM remodeling during glaucoma progression. In this study, we examined miRNA expression profiles of ONHA exposed to cyclic stretch. Methods: Primary human normal ONHA cell strains (n=3) were exposed to 0-12% cyclic stretch for 24 hours. miRNA PCR arrays were used to determine expression changes in profibrotic and anti-fibrotic miRNAs. Results: We found that specific miRNAs were consistently dysregulated across three independent strains of ONHA. Statistical significance could not be detected, but these patterns may represent biologically meaningful changes. Conclusion: Stretch modulates miRNA expression in cultured human ONHA and may be responsible for ECM alterations at the LC. Dysregulated miRNAs may serve as novel targets or models for future therapeutics.Item miRNA Profiling of Optic Nerve Head Astrocytes Exposed to Cyclic Stretch(2022) Rangan, Rajiv; Tovar-Vidales, TaraIntroduction: Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, a leading cause of irreversible blindness consequent to retinal ganglion cell (RGC) degeneration. Elevated IOP induces biomechanical aberrations within ocular tissues - including the transmission of biomechanical stretch through the lamina cribrosa (LC) region of the optic nerve head (ONH), the site where RGC axon damage first occurs. LC cells and ONH astrocytes (ONHA), the primary cells of the LC, respond to stretch in a manner that promotes pathological extracellular matrix (ECM) remodeling (fibrosis) and mechanical damage of RGCs within the ONH. A complex set of molecular mechanisms regulate ECM remodeling. Part of this regulation may involve microRNAs (miRNAs), small molecules that can inhibit protein expression by binding to and silencing mRNA. In this study, we examined miRNA expression profiles of ONHA exposed to cyclic stretch. We hypothesized that cyclic stretch would induce upregulation of miRNAs that silence anti-fibrotic protein translation and downregulation of miRNAs that silence pro-fibrotic protein translation, promoting a net-fibrotic molecular signaling environment. Methods: Primary human normal ONHA cell strains (n=3) were exposed to 0-12% cyclic stretch for 24 hours; controls were exposed to 0% stretch. RNA samples were collected from stretched and control cells, and miRNA PCR arrays were used to determine expression changes for miRNAs associated with fibrosis. Expression fold changes were normalized to SNORD68. The bioinformatics tool TargetScan was used to predict mRNA targets for any dysregulated miRNAs. Induction of fibrotic cellular changes by cyclic stretch was confirmed by western blotting of conditioned media for secreted proteins. Results: miR-146b-5p was found to be significantly upregulated by +5.97-fold (P = 0.029) in stretched ONHA. Predicted mRNA targets for miR-146b-5p are known to be involved in fibrosis and cell survival, among other functions. Preliminary data indicates upregulation of secreted proteins associated with fibrosis (TGFβ2, Fibronectin, Transglutaminase 2) by stretched ONHA. Conclusions: Stretch modulates miRNA expression in cultured human ONHA, miR-146b may mediate ECM alterations and other pathological changes at the LC. Future experimental directions will include assessing co-expression of other miR-146 family miRNAs, validating putative mRNA targets and elucidating the mechanisms by which specific miRNA and their targets modulate ECM remodeling.Item Modulation of Mitochondrial Metabolic Parameters and Antioxidant Enzymes in Healthy and Glaucomatous Trabecular Meshwork Cells with Hybrid Small Molecule SA-2(MDPI, 2023-07-29) Amankwa, Charles E.; Young, Olivia; DebNath, Biddut; Gondi, Sudershan R.; Rangan, Rajiv; Ellis, Dorette Z.; Zode, Gulab S.; Stankowska, Dorota L.; Acharya, SuchismitaOxidative stress (OS)-induced mitochondrial damage is a risk factor for primary open-angle glaucoma (POAG). Mitochondria-targeted novel antioxidant therapies could unearth promising drug candidates for the management of POAG. Previously, our dual-acting hybrid molecule SA-2 with nitric oxide-donating and antioxidant activity reduced intraocular pressure and improved aqueous humor outflow in rodent eyes. Here, we examined the mechanistic role of SA-2 in trabecular meshwork (TM) cells in vitro and measured the activity of intracellular antioxidant enzymes during OS. Primary human TM cells isolated from normal (hNTM) or glaucomatous (hGTM) post-mortem donors and transformed glaucomatous TM cells (GTM-3) were used for in vitro assays. We examined the effect of SA-2 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro using Seahorse Analyzer with or without the oxidant, tert-butyl hydroperoxide (TBHP) treatment. Concentrations of total antioxidant enzymes, catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GPx) were measured. We observed significant protection of both hNTM and hGTM cells from TBHP-induced cell death by SA-2. Antioxidant enzymes were elevated in SA-2-treated cells compared to TBHP-treated cells. In addition, SA-2 demonstrated an increase in mitochondrial metabolic parameters. Altogether, SA-2 protected both normal and glaucomatous TM cells from OS via increasing mitochondrial energy parameters and the activity of antioxidant enzymes.